FASN Antibody

What is FASN and why is it an important research target?

Fatty Acid Synthase (FASN) is a multifunctional enzyme encoded by the FASN gene that catalyzes the de novo biosynthesis of long-chain saturated fatty acids. In humans, the canonical protein has 2511 amino acid residues with a molecular mass of approximately 273.4 kDa . FASN is primarily localized in the cytoplasm and is ubiquitously expressed across many tissue types, with prominent expression in brain, lung, and liver . It plays critical roles in fatty acid metabolism and inflammatory response pathways. FASN has gained significant research attention due to its overexpression in various human carcinomas, including breast, lung, and prostate cancers, where its expression is often associated with poor prognosis . Additionally, FASN has emerged as a potential target for both cancer diagnosis and treatment, as well as for metabolic syndrome .

What applications are FASN antibodies commonly used for in research?

FASN antibodies are utilized across multiple research applications:

• Western Blotting (WB): Detection of FASN protein expression with expected band size at ~273 kDa

• Immunohistochemistry (IHC): For paraffin-embedded and frozen tissue sections

• Immunocytochemistry/Immunofluorescence (ICC/IF): Visualization of cytoplasmic FASN expression in cells like MCF7

• Immunoprecipitation (IP): For protein complex isolation and interaction studies

• ELISA: Quantification of FASN in biological samples

Most commercially available antibodies have been validated for specific applications with recommended dilutions (e.g., 1:1000 for WB, 1:500 for IHC) .

How do I select the appropriate FASN antibody for my specific research application?

Selection should be based on:

• Target application: Verify the antibody has been validated for your specific application (WB, IHC, IF, etc.)

• Species reactivity: Confirm the antibody recognizes FASN in your experimental species (human, mouse, rat, etc.)

• Clonality: Monoclonal antibodies provide consistent results with high specificity to a single epitope, while polyclonal antibodies may offer higher sensitivity by recognizing multiple epitopes

• Epitope region: Consider whether you need an antibody targeting a specific domain of FASN (e.g., thioesterase domain or β-ketoacyl synthase domain)

• Validation data: Review literature citations and validation data (knockout cell lines, peptide competition) to ensure specificity

• Format: Determine if you need unconjugated or conjugated antibodies for specific detection methods

What are the recommended protocols for FASN detection by Western blot?

For optimal Western blot detection of FASN:

• Sample preparation:

  • Use appropriate lysis buffers (e.g., RIPA) with protease inhibitors

  • Due to FASN's high molecular weight (273 kDa), use lower percentage gels (6-8%)

  • Load 10-30 μg of total protein per lane

• Electrophoresis conditions:

  • Run at lower voltage (80-100V) for better resolution of high molecular weight proteins

  • Extend transfer time (overnight at 30V is recommended) for complete transfer

• Antibody conditions:

  • Primary antibody: Use at 1:1000 dilution in 5% BSA or milk

  • Longer primary antibody incubation (overnight at 4°C) improves signal

  • Secondary antibody: HRP-conjugated at 1:2000-1:5000 dilution

• Controls:

  • Positive control: HepG2 or MCF7 cell lysates (high FASN expression)

  • Negative control: FASN knockout cell lines for specificity validation

• Detection:

  • Expected band size: ~273 kDa (main band)

  • May occasionally see minor cross-reacting lower MW bands in liver tissue

How can I optimize immunohistochemistry protocols for FASN detection in tissue samples?

For optimal IHC detection of FASN:

• Tissue preparation:

  • Formalin-fixed paraffin-embedded (FFPE) sections: 4-6 μm thickness

  • Antigen retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 20 minutes

• Blocking and antibody conditions:

  • Block with 5-10% normal serum (matching species of secondary antibody)

  • Primary antibody: 1:500 dilution, incubate overnight at 4°C

  • Secondary antibody: Use polymer-based detection systems for enhanced sensitivity

• Controls and validation:

  • Positive control: Adipocytes (FASN is a marker for adipocytes)

  • Negative control: Omit primary antibody or use isotype control

  • Pattern: Expect cytoplasmic staining pattern

• Counterstaining:

  • Use hematoxylin for nuclear counterstaining

  • Avoid overstaining which may mask specific FASN signals

• Optimization tips:

  • Titrate antibody concentration for optimal signal-to-noise ratio

  • For weakly expressing samples, consider signal amplification methods

What methods can be used to measure FASN enzymatic activity in biological samples?

The preferred method for measuring FASN activity is the NADPH absorbance assay:

• NADPH absorbance assay principle:

  • FASN catalyzes the conversion of acetyl-CoA and malonyl-CoA to palmitate

  • This reaction consumes NADPH, which can be monitored spectrophotometrically

  • The decrease in absorbance at 340 nm corresponds to NADPH consumption and FASN activity

• Protocol overview:

  • Prepare reaction mixture containing substrate (acetyl-CoA, malonyl-CoA)

  • Add NADPH and sample containing FASN

  • Monitor decrease in absorbance at 340 nm over time

  • Calculate activity based on the rate of NADPH consumption

• Considerations:

  • Maintain temperature at 37°C during measurement

  • Include appropriate controls (positive control, no enzyme control)

  • Normalize activity to protein concentration

  • Ensure linear range of the assay

• Alternative methods:

  • Radiometric assays using [14C]-labeled substrates

  • Mass spectrometry-based methods to directly measure palmitate production

How can FASN antibodies be used to study post-translational modifications of FASN?

FASN undergoes various post-translational modifications (PTMs) that affect its activity and function, particularly acetylation:

• Antibody-based enrichment for acetylation site identification:

  • Immunoprecipitate FASN using specific antibodies (1:100 dilution recommended)

  • Process samples for mass spectrometry analysis

  • Identify acetylated lysine residues by MS/MS

• Protocol workflow:

  • Cell/tissue lysis under conditions that preserve PTMs

  • FASN immunoprecipitation using specific antibodies

  • SDS-PAGE separation and in-gel digestion

  • MS analysis for PTM identification

• Validation approaches:

  • Compare identified acetylation sites with databases like Protein Lysine Modification Database (PLMD) and PhosphoSitePlus®

  • Use site-specific antibodies if available

  • Employ mutagenesis to confirm functional significance

• Key findings:

  • Eight acetylated lysine residues have been identified in FASN from L3 stage larvae

  • Acetylation can affect FASN enzymatic activity and stability

What is the role of FASN in cancer immune evasion and how can antibodies help study this process?

FASN plays a critical role in cancer immune evasion through multiple mechanisms:

• FASN expression and immune landscape correlation:

  • FASN expression negatively correlates with infiltrating immune cells associated with cancer suppression

  • High FASN expression is associated with reduced cytolytic activity signatures and decreased HLA-I expression

  • Bioinformatic analysis of TCGA data shows FASN overexpression in "wound healing" immune subtype C1 tumors

• Experimental approaches using antibodies:

  • Analyze FASN expression in tumor samples by IHC and correlate with immune cell infiltration

  • Use FASN antibodies to detect changes in expression following immune challenge

  • Study FASN-regulated pathways through co-immunoprecipitation experiments

• Functional studies:

  • CRISPR/Cas9-based FASN knockout cancer cells show enhanced susceptibility to T-cell-mediated killing

  • FASN depletion results in reduced mitochondrial OXPHOS and downregulation of electron transport chain complexes

  • FASN blockade reverses immunosuppressive features of "cold" tumors to a "hot" immunostimulatory context

• Therapeutic implications:

  • FASN inhibition may sensitize tumors to immune checkpoint inhibitors

  • Combining FASN antibodies with other immune markers can identify patients likely to respond to immunotherapy

How can researchers develop and validate new FASN-specific monoclonal antibodies for specialized applications?

Development and validation of new FASN monoclonal antibodies involves:

• Antigen design and antibody generation:

  • Select immunogenic epitopes from different FASN domains (e.g., Ser10-Cys212 region used successfully)

  • Consider recombinant protein fragments or synthetic peptides as immunogens

  • Use hybridoma technology for monoclonal antibody production

• Screening and initial characterization:

  • ELISA screening against immunizing antigen

  • Evaluate cross-reactivity with FASN orthologs (human, mouse, rat)

  • Test in preliminary applications (WB, IHC)

• Validation strategies:

  • Specificity testing using FASN knockout cell lines

  • Western blot showing single band at expected molecular weight (~273 kDa)

  • Immunoprecipitation followed by mass spectrometry confirmation

  • Testing across multiple cell lines with varying FASN expression levels

• Advanced validation:

  • Epitope mapping to confirm binding site

  • Sandwich ELISA development using paired monoclonal antibodies

  • Competitive binding assays with established antibodies

• Application optimization:

  • Detailed titration for each application

  • Testing in multiple experimental conditions

  • Cross-validation with existing antibodies

What are common issues in FASN detection by Western blot and how can they be resolved?

Common issues and solutions for FASN Western blot detection:

• Weak or no signal:

  • Problem: Insufficient protein transfer due to high molecular weight (273 kDa)

  • Solution: Use lower percentage gels (6-8%), increase transfer time, or employ specialized transfer systems for high molecular weight proteins

• Multiple bands:

  • Problem: Degradation products or cross-reactivity

  • Solution: Use fresh samples with protease inhibitors; may normally see minor cross-reacting lower MW bands in liver tissue

• High background:

  • Problem: Non-specific binding or insufficient blocking

  • Solution: Increase blocking time/concentration, reduce antibody concentration, use more stringent washing

• Inconsistent results:

  • Problem: Variability in FASN expression or sample preparation

  • Solution: Include positive controls (HepG2 or MCF7 cell lysates) and loading controls

• Size discrepancies:

  • Problem: Observed molecular weight differs from expected 273 kDa

  • Solution: Consider post-translational modifications, use molecular weight markers suitable for high MW range

How should researchers interpret FASN expression data in the context of cancer research?

Interpretation of FASN expression data in cancer research:

• Expression level considerations:

  • FASN overexpression is common in most human carcinomas

  • In breast cancer, FASN levels directly correlate with tumor size

  • Expression is an indicator of poor prognosis in breast and prostate cancers

• Correlation with immune parameters:

  • FASN expression negatively correlates with infiltrating immune cells

  • High FASN expression associates with reduced cytolytic activity and HLA-I expression

  • FASN expression anticorrelates with signatures of T-cell accumulation and IFNγ-mediated response to anti-PD1 therapy

• Functional implications:

  • High FASN expression indicates metabolic reprogramming toward de novo lipogenesis

  • May suggest potential resistance to immunotherapy

  • Could indicate sensitivity to FASN inhibitors

• Technical considerations:

  • Compare results across multiple detection methods (WB, IHC, qPCR)

  • Validate with appropriate controls and normalization

  • Consider tissue/cellular context for proper interpretation

How can researchers address contradictory data when studying FASN in different experimental systems?

Strategies for addressing contradictory FASN data:

• Methodological considerations:

  • Different antibodies may recognize distinct epitopes, yielding different results

  • A monoclonal-monoclonal ELISA can show different pattern of FASN levels compared to polyclonal-monoclonal ELISA

  • Use multiple antibodies targeting different FASN domains to confirm findings

• Biological variables:

  • FASN expression and function can vary across cell types and tissues

  • Consider post-translational modifications that may affect antibody binding

  • Account for species differences in FASN sequence and regulation

• Experimental design factors:

  • Standardize sample collection, processing, and storage protocols

  • Use consistent experimental conditions (culture conditions, treatment durations)

  • Include appropriate positive and negative controls

• Integration approaches:

  • Combine multiple techniques (WB, IHC, qPCR, activity assays)

  • Supplement antibody-based detection with functional assays

  • Correlate with genomic or transcriptomic data when available

• Validation strategies:

  • Validate key findings with genetic approaches (siRNA, CRISPR/Cas9)

  • Perform rescue experiments to confirm specificity

  • Replicate in independent experimental systems

How can FASN antibodies be used to study the role of FASN in immunotherapy resistance mechanisms?

FASN antibodies can help elucidate immunotherapy resistance mechanisms:

• Profiling FASN expression in responders vs. non-responders:

  • IHC analysis of tumor biopsies before and after immunotherapy

  • Correlation of FASN expression with response to immune checkpoint inhibitors

  • FASN mutations that inactivate lipogenic function correlate with improved response to ICIs

• Mechanistic studies:

  • Detect FASN-driven PD-L1 palmitoylation, a critical post-translational modification for cell membrane-bound PD-L1 functionality

  • Evaluate FASN influence on MHC-II expression and antigen presentation

  • Assess FASN impact on tumor microenvironment composition

• Therapeutic strategies:

  • Monitor FASN expression changes following combination therapy (FASN inhibitors + immunotherapy)

  • Validate FASN as biomarker for patient stratification

  • Develop FASN-targeted approaches to enhance immunotherapy efficacy

• Technical approaches:

  • Multiplex immunofluorescence to simultaneously detect FASN and immune markers

  • Live cell imaging to track FASN-immune cell interactions

  • Single-cell analysis to identify FASN expression in resistant cell populations

What are the current methodologies for studying FASN interactions with other proteins in the tumor microenvironment?

Methodologies for studying FASN protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use FASN antibodies (recommended dilution 1:100) to pull down protein complexes

  • Identify interacting partners by Western blot or mass spectrometry

  • Can be performed under native conditions to preserve physiological interactions

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions in fixed cells or tissues

  • Uses pairs of antibodies against FASN and potential interacting partners

  • Visualize interactions as fluorescent spots via microscopy

• FRET/BRET approaches:

  • Tag FASN and interacting proteins with compatible fluorophores/luminescent proteins

  • Measure energy transfer to detect close proximity in living cells

  • Can provide temporal information about dynamic interactions

• Yeast two-hybrid and mammalian two-hybrid:

  • Screen for novel FASN interacting partners

  • Validate interactions in relevant cellular contexts

  • Map interaction domains

• Cross-linking mass spectrometry:

  • Stabilize transient interactions through chemical cross-linking

  • Enrich FASN complexes using specific antibodies

  • Identify interacting regions at amino acid resolution

How can researchers develop assays to evaluate FASN inhibitors as potential immunomodulatory agents?

Developing assays for evaluating FASN inhibitors as immunomodulators:

• FASN activity assays:

  • NADPH absorbance-based assays to confirm target engagement

  • Measure palmitate production using mass spectrometry

  • Track cellular lipid composition changes using lipidomics

• Immunological readouts:

  • Analyze T cell infiltration and activation in FASN-inhibited tumors

  • Measure changes in HLA-I and HLA-II expression following FASN inhibition

  • Assess cytolytic activity against FASN-inhibited cancer cells

• In vitro co-culture systems:

  • Co-culture FASN-inhibited cancer cells with immune cells

  • Use impedance-based real-time monitoring of cytolytic responses

  • Measure immune checkpoint expression (PD-L1) and T cell function markers

• In vivo models:

  • Combine FASN inhibitors with immune checkpoint inhibitors in tumor models

  • Track changes in tumor microenvironment composition

  • Monitor adaptive immune responses in FASN-inhibited settings

• Translational approaches:

  • Develop FASN expression/activity as companion biomarker for immunotherapy

  • Establish FASN-immune signatures to predict response to combination therapy

  • Design rational sequential or combination treatment strategies